1. Field of the Invention
This invention relates to methods for the inactivation of viruses in virus-contaminated pharmaceutical compositions containing proteinaceous components, such as coagulation factors, by the use of chemical inactivation and/or physical processes. In particular, this invention relates to blood plasma or other plasma protein-containing compositions which are to be rendered free of viral infectivity, such blood plasma or fractions thereof having valuable labile proteins, such as, for example Factor IX. .A specific method of the invention requires the use of sodium thiocyanate and ultrafiltration.
2. Description of Related Art
Blood is made up of solids (cells, i.e., erythrocytes, leukocytes, and thrombocytes) and liquid (plasma). The cells contain potentially valuable substances such as hemoglobin, and they can be induced to make other potentially valuable substances such as interferons, growth factors, and other biological response modifiers. The plasma is composed mainly of water, salts, lipids and proteins. Prior to the availability of a more detailed description of individual protein components, the proteins were divided into groups; initially as simply "albumins" and "globulins". Typical antibodies (immune serum globulins) found in human blood plasma include those directed against infectious hepatitis, influenza H, etc.
Whole blood must be carefully typed and cross matched prior to administration in blood transfusions. Plasma, however, does not generally require prior testing. For certain applications, only a proper fraction of the plasma is required, such as factor VIII complex for treatment of hemophilia or von Willebrand's disease. The rationale for use of specific fractions of blood is that blood contains a number of differently formed elements as well as various plasma proteins and constituents which have many functions. Thus, a single donation of a unit of whole blood can provide red blood cells, platelets, plasma, and cryoprecipitated factor VIII-fibrinogen concentrate. Pheresis procedures are able to supply large quantities of granulocytes, platelets, and plasma from single donors. The rationale for using blood components is that a patient usually requires replacement of only a specified component (See: Greenwalt et al: General Principles of Blood Transfusion, A.M.A. Editorial Board, 1978). Remaining components can be then used to treat patients who require other specific components, thereby allowing several patients to benefit from each blood unit donated, thereby maximizing the benefit realizable therefrom. The separation of blood into components and their subsequent fractionation allows the proteins to be concentrated. Of great importance, too, is the fact that the plasma fractions can be stored for much longer periods than whole blood and they can be distributed in liquid, frozen, or dried state. Finally, it allows, in some cases, salvaging from blood banks the plasma portions of outdated whole blood that are unsafe for administration as whole blood.
Proteins found in human plasma include prealbumin, retinol-binding protein, albumin, alpha-globulins, beta-globulins, the coagulation proteins (Factors II, VII, IX, X, V, VIII, XI, XII, XIII and inhibitors such as protein C, antithrombin III, etc.) fibronectin, immunoglobins (immunoglobulins G, A, M, D, and E), and the complement components. There are currently more than 100 plasma proteins that have been described. A comprehensive listing can be found in the "The Plasma Proteins", ed. Putnam, F. W., Academic Press, New York (1975)
Proteins found in the blood cell fraction include hemoglobin, fibronectin, fibrinogen, enzymes of carbohydrate and protein metabolism, etc. In addition the synthesis of other proteins can be induced, such as interferons and growth factors.
Plasma can be chemically fractionated to provide albumin or plasma protein fraction, Factor VIII concentrate, Factor IX complex and immune serum globulin.
Blood plasma fractionation generally involves the use of organic solvents such as ethanol, ether and polyethylene glycol at low temperatures and at controlled pH values to effect precipitation of a particular fraction containing one or more plasma proteins. The resultant supernatant can itself then be precipitated and so on until the desired degree of fractionation is attained. More recently, separations are based on chromatographic processes. A survey of blood fractionation appears in Kirk-Othmer's Encyclopedia of Chemical Technology, Third Edition, Interscience Publishers, Volume 4, pages 25 to 62.
The major components of a cold ethanolfractionation are as follows:
______________________________________ Fraction Proteins ______________________________________ I fibrinogen; cold insoluble globulin; Factor VIII; properdin II and III IgG; IgM; IgA; fibrinogen; beta-lipoprotein; prothrombin; plasminogen; plasmin inhibitor; Factor V; Factor VII; Factor IX; Factor X; thrombin; antithrombin; isoagglutinins; ceruloplasmin; complement C'1, C'3 IV-1 alpha-1-lipoprotein, ceruloplasmin, plasmin-inhibitor; Factor IX; peptidase; alpha-and-beta-globulins IV-4 transferrin; thyroxine binding globulin; serum esterase; alpha-1-lipoprotein; albumin; alkaline phosphatase V albumin; alpha-globulin VI alpha-1-acid glycoprotein; albumin ______________________________________
The above fractionation scheme can serve as a basis for further fractionations. Fraction II and III, for example, can be further fractionated to obtain immune serum globulin (ISG), a mixture primarly of Igb antibodies.
Another fractionation scheme involves use of frozen plasma which is thawed into a cryoprecipitate containing AHF (antihemophilic factor) and fibronectin and a cryosupernatant. The cryoprecipitate is then fractionated into fibronectin and AHF.
Polyethylene glycol is among the agents which have been used to prepare high purity AHF and non-aggregated ISG.
In the development of new products from human plasma, at least three major problems are always present. These are contamination with pyrogens (endotoxins), transmission of viral hepatitis or other viral diseases, and activation of the coagulation enzymes.
Solutions of pharmaceutical compositions which are intended to be parenterally administered in man (or in veterinary applications) are required to be sterilized from infective microorganism such as bacteria and fungi. A common method is to subject the composition to steam sterilization (autoclaving) at temperatures in excess of 100.degree. C. at hyperbaric pressures for a time sufficient to be efficacious. This treatment kills viruses but can be deleterious or destructive to certain heat-sensitive compositions such as those which contain proteinaceous components such as coagulation Factors VIII, IX, II, VII, X, and the like.
Pyrogens are lipopolysaccharides (LPS) derived from the outer cell wall of gram-negative bacteria. They are toxic materials which are also known as endotoxins to distinguish them from toxic substances synthesized and excreted by the intact bacterium. Pyrogens have numerous biologic activities which include the production of fever, activation of clotting mechanisms and induction of shock. Consequently, in addition to the need for sterility from infectious agents, it is essential that pyrogenic substances be removed and that the causative bacteria be rendered innocuous by sterilization or other such treatment of the final plasma product.
Blood coagulation factors play a vital role in the normal coagulation mechanism. For instance, patients with a deficiency of Factor IX exhibit severe bleeding problems ("Hemophilia B"). It would be desirable to be able to isolate substantial quantities of Factor IX and other vitamin K-dependent proteins for therapeutic administration, as well as for scientific study.
Factor IX complex is a lyophilized pooled plasma derivative rich in Factors IV, VII, IX and X. It is an alternative to plasma therapy. It supplies vitamin K-dependent clotting factors in a much smaller volume than plasma but with a significantly higher hepatitis risk.
Factor IX containing concentrates are a unique and highly valuable blood product which are life-saving when used to control bleeding in patients suffering with Factor IX deficiency (Hemophilia B). These products have also been used to treat those patients afflicted with Hemophilia A having inhibitors, although clinical verification of this application is still in progress. Factor IX containing concentrates are also used to arrest serious hemorrhages or to avert operative and post operative bleeding in patients with other congenital clotting factor deficiencies and for multiple factor deficiency induced by an overdose of warfarin-type drugs, i.e., oral anticoagulants.
Commercial concentrates of Factor IX have been previously prepared using ion exchange resins to bind vitamin K-dependent clotting factors and separate these proteins from the bulk of other plasma proteins. These clotting factor concentrates are then eluted from the resin and vialed for therapeutic use without further purification. Such concentrates tend to have thrombogenic potential probably because they contain extraneous vitamin K-dependent clotting factors and/or phospholipid. Further, such concentrates have been a suspected vehicle in the transmission of viral diseases including hepatitis and acquired immune deficiency syndrome ("AIDS"). Further, crude concentrates of Factor IX are not stable for long periods in solution and therefore cannot be used for constant infusion therapy which limits their value in chronic replacement therapy.
Recent efforts to create Factor IX using a recombinant DNA approach have been frustrated by the difficulty encountered in separating Factor IX from culture supernatants with currently accepted techniques. (See: Anson DS, Austen DEG, and Brownless GG. "Expression of active human clotting Factor IX from recombinant DNA clones in mammalian cells." Nature 1985; 315:683-685; de la Salle H. Altenburger W. Elkaim R., Dott K., et al. "Active gamma-carboxylated human Factor IX expressed using recombinant DNA techniques." Nature 1985; 316:268-270; and Busby S., Kumar A., Joseph M., Halfpap L. Insley M. et al. "Expression of active human Factor IX in transfected cells." Nature 1985; 316:271-273). Thus, there remains a medical need for a safe preparation of Factor IX obtained from human plasma.
Numerous attempts have been made to inactivate viruses such as lipid-containing viruses of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in mammalian, especially, human, blood plasma. It is the practice in some countries to effect inactivation of the hepatitis B virus in the blood plasma by contacting the plasma with a viral inactivating agent of the type which crosslinks with the proteinaceous protein of hepatitis B virus or which interacts with the nucleic acid of the virus. For instance, it is known to attempt to inactivate hepatitis B virus by contact with an aldehyde (such as formaldehyde) whereby crosslinking to the protein is effected and the hepatitis B virus is inactivated. It is also known to effect inactivation of the virus by contact with beta-propiolactone (BPL), an agent which acts on the nucleic acid as well as protein components of the virus. It is further known to use ultraviolet (UV) light, especially after a beta-propiolactone treatment. It is believed that these methods are not suitable for the inactivation of the virus in plasma due to the observation that most of these inactivating agents (formaldehyde, beta-propiolactone and sodium hypochlorite) denatured or altered the valuable proteinaceous components of the plasma, especially so-called "labile" blood coagulation factors of the plasma.
The removal of bacteria and fungi from such heat-sensitive proteinaceous compositions is generally accomplished by the use of a bacterially retentive filter. Typical examples are the membrane filters in the porosity range 0.1-0.2 microns (100-200 nanometers) produced by Pall Corporation and Millipore Corporation. Generally, the proteinaceous components in the pharmaceutical composition remain undamaged. However, it is known that membrane filters can fail to retain highly infectious and dangerous microorganisms such as virus particles. Filter devices can be designed to retain some virus particles if the effective filter porosity is of a small enough size. Such devices have sometimes been used to harvest viral particles, e.g., during the manufacture of viral vaccines. Most viral particles, however, are smaller in size than the effective porosity of the membrane filter and are not retained. For example, the hepatitis B virus, which may be present in coagulation factor solutions made from human plasma, has a diameter of 42 nanometers (nm) and will readily pass through a 100 nm (0.1 micron) membrane filter.
It is well-known that plasma and products made from plasma may transmit hepatitis. Initially, interest in viral transmission focused primarily on hepatitis B antigen (HB.sub.s A.sub.g) as an indicator of the presence of the offending agent (hepatitis B virus) and attempts at eliminating this agent have led to widespread screening of all plasma used in transfusion by commercially available and approved laboratory procedures. While such laboratory screening has apparently decreased the incidence of hepatitis B in patients receiving whole blood transfusions, there has not been significant improvement in the incidence of the disease transmitted from plasma products Chronic users of blood products are at risk from the disease unless maintained in an immune state by innoculation with a vaccine. While effective, this may have other clinical risks associated with it. Attempts to remove the virus by various adsorption procedures or precipitation techniques, e.g. with polyethylene glycol, have not proven to fully eliminate infectivity. There is some evidence that the combination of ultraviolet light and B-propionolactone may be helpful in inactivating the virus in certain plasma products. However, there is some apprehension the B-propionolactone has carcinogenic properties.
In order to increase the safety of pharmaceutical compositions which contain heat-sensitive proteinaceous components and which may contain dangerous virus particles, additional processing is required. This processing includes heating solutions of the proteinaceous components with special stabilizers to protect biologic potency, heating lyophilized preparations of the proteinaceous components, and treating solutions of proteinaceous components with organic solvents and other virucidal agents. Most of these methods are burdensome, time consuming, or destructive of the protein due to the rigorousness of the treatment. There are still questions about the efficacy of any one of these procedures applied singly to plasma products.
While the development of screening tests for hepatitis B has been of limited value in reducing transmission of the disease, the identification of this virus (as well as the hepatitis A virus) has led to the recognition of a third virus which is apparently responsible for the majority of cases of hepatitis transmitted by blood plasma derivatives. This virus is referred to as "non-A, non-B hepatitis".
Methods for the inactivation of hepatitis B virus in the plasma are known but are usually impractical. One method involves the addition of antibodies to the plasma whereby an immune complex is formed. The expense of antibody formation and purification add significantly to the cost of the plasma production; furthermore, there is no assurance that a sufficient quantity of non-A, non-B virus is inactivated since the method is specific for the hepatitis B virus. There is currently no approved and available test for non-A, non-B antibodies or virus, though there are reports of progress in achieving this; hence, it is not as yet possible to select plasma containing high titers of anti non-A, non-B antibody, nor indicate that this approach would be practicable.
As progress has been made in the development of human plasma derived therapeutics, the necessity of viral sterilization has become manifest. Stable plasma protein solutions can withstand pasteurization but labile blood coagulation factors are most often inactivated or significantly reduced in potency during such heating. This restricts practical applications. As a result, recipients of Factor VIII, gamma-globulin, Factor IX, fibrinogen, etc., must often accept the risk that the valuable protein components being administered may be contaminated with hepatitis viruses as well as other infectious viruses. As a result, these recipients face the danger of becoming infected by these viruses and having to endure the damage which the virus causes to organ systems and consequent incapacitation and illness which may lead to death. Therefore, there is a need for a more effective yet practicable method for purification of the heat-sensitive plasma, specifically a method for viral sterilization without the use of heat.
Thus a very special need exists for the development of means and methods for the manufacture and isolation of highly purified Factor IX which can thereafter be formulated into a potent, quick-acting, therapeutic blood product which is stable in vitro and which provides effective relief for patients encountering a critical bleeding incident.
The present invention is directed to achieving three goals, namely, (1) a safe, 2) viral inactivated protein-containing composition, (3) without incurring substantial protein denaturation. These three goals are not necessarily compatible since, for example sterilization inactivates viral infectivity, but substantially denatures the valuable plasma proteins, for example, beta-propiolactone inactivates viral infectivity but is unsafe, and substances such as formaldehyde inactivate viruses, but also substantially denaturate the valuable plasma proteins, for example, Factor IX.
It is therefore desirable to provide a process for obtaining protein-containing compositions which does not substantially denature the valuable protein components therein and which does not entail the use of proven carcinogenic agents (such as B-proprionolactone). More especially, it is desirable to provide blood protein, containing compositions in which substantially all of the hepatitis viruses and other viruses present are inactivated. It is a further object to provide products from cancer or normal cells or from fermentation processes of cells following given insertion of recombinant DNA which are substantially free of virus, including lipid-containing viruses, which comprise a category containing known infective agents of plasma.